http://teachline.ls.huji.ac.il/~92984/
From Transgene to Protein, Station
IVb
T. Bruck, Dr. E.R. Bennett, E. Podoly
Introduction
Methods• Experimental
•
Computational
• Interactome• Driving
Forces• Energy
Proteins
Proteins rarely function in isolation. Protein-Protein interactions
(PPIs) are an integral part of cellular processes and functions.
Understanding and characterization
of data coming from proteomic
experiments is one of the major
challenges of the post-genomic era:
This is the time of the InteractomeInteractome.Genomics > Proteomics > Interactomics
The Post-Genomic Era
Interactomics is a study of interaction
network, aimed at mapping the molecular
interactions in cells using computational
and experimental methods.
Interactome – The Protein’s Social Utility
proteins
The Driving Forces of Interactions
· Disulfide bonds
· Hydrophobic
· Hydrogen bonds
· Electrostatic
I. Covalent Interactions
II. Non-Covalent Interactions
Covalent bonds result from sharing pairs of electrons between atoms.
Disulfide bonds are formed
between the thiol groups of cysteine
residues.
The Driving Forces of Interactions
Non-covalent bonds involve dispersed variations of electromagnetic interactions:
Hydrophobic interactions are formed between non-polar
atoms.
Hydrogen bonds are formed between an electronegative atom
and a hydrogen atom bonded to another electronegative atom.
Electrostatic interactions are formed between charged atoms.
Bond Strength (kcal/mole)
Hydrophobic interactions 1-2
H-bonds 3-7
Ionic bonds 10
Disulfide bridge 51
Energy of Interactions
Noncovalent interactions are weak as
compared with a covalent bond, but:
1. They work jointly to hold two
interacting surfaces together.
2. A surface topography enables
substantial areas of two interacting
surfaces to approach each other closely.
Methods to Study PPIs
Tracking PPIs and delineating their detailed information are both
interest for researchers in many fields. Multitude of methods detect
PPIs, each has its own strengths and weaknesses.
Specificity: most of the interactions
detected occur in reality.
Sensitivity: many interactions that
occur in reality are detected.
Two-Hybrid System (Y2H)
A genetic method based on transcription factor structure in yeast.
A functional transcription factor consists of two separable domains:
DNA binding domain (BD)
Activation domain (AD)
In Y2H system, these two
domains are separated and each is
fused to the protein of interest (X
and Y, respectively).
Physical interaction between BD-X and AD-Y reconstitutes the
transcription factor that activates the transcription of reporter gene.
Monitoring interactions of two proteins, one of them is labeled with
a donor and the other with an acceptor, by following the energy
transfer between them.
Fluorescence Resonance Energy Transfer*
* Termed also: "Förster Resonance Energy Transfer“, after the German scientist Theodor Förster
Protein A is labeled with CFP
while protein B is labeled with
YFP.
If the A and B are adjacent to each
other, an energy transfer happens.
Co - ImmunoPrecipitation
An antibody specific to protein X is added to a cell lysate.
Identification of proteins in the pellet can be determined by western
blot/sequencing a purified protein band/antibody against protein Y.
The antibody-protein complex is pelleted with protein-G sepharose.
Unbound protein are washed.
Size Exclusion Chromatography*
* Termed also: Gel Filtration Chromatography
Porous beads
separate different
sizes of
molecules.Small proteins
enter the pores
and have a
longer path and
longer transist
time than bigger
proteins.
Size Exclusion Chromatography
Protein Complexes will
have even shorter path and
transist time compared to
the its protein components.
Affinity Chromatography
A tagged protein of interest
binds to beads coated with
antibodies against its tag.
A cell lysate is passed
through the affinity
column.
Beads coated with
antibodies that
bind their antigen.
Affinity Chromatography
Binding partners are not eluted.
Tag can be broken by an
enzyme, following by proteins’
identification as before.
SPR reflectivity measures the changes in the local index of
refraction upon adsorption of the target molecule to the metal
surface.
Surface Plasmon Resonance*
* Termed also: Dual Polarisation Interferometry
Comparison of Methods
Y2H
FRET
Co-IP
Size Ch.
Affinity Ch.
Fluorescence
SPR
Crosslinking
Sensitivity Specificity StrengthsTag/Ig
High Low
Immobilization dependent
C/E/P
+/-/-
+/+/+
+/+/+
+/+/+
-/-/-
+/-
+/+/+
-/+
-/-
+/+/+ +/-Kinetics
Equilibrium
Simplicity
Preparative
Screening+/+
High
High
High
Med.
Med.
Weaknesses
False pos.
Apparatus
High
High
High
Kinetics Equilibrium
Kinetics Equilibrium
Crosslinker dependent
Med.
Med. High
Screening In vivo
High
T.C Apparatus
Time Consuming
Time Consuming
Time Consuming
Resource Comments
BIND Biomolecular Interaction Network Database at the University of Toronto, Canada.
CYGD PPI section of the Comprehensive Yeast Genome Database.
DIP Database of Interacting Proteins at UCLA. No species restriction.
GRID General Repository for Interaction Datasets. Mount Sinai Hospital, Toronto, Canada
HPRD The Human Protein Reference Database. Institute of Bioinformatics, Bangalore, India & Johns Hopkins University, Baltimore, MD, USA.
HPID Human Protein Interaction Database. Department of computer Science and Information Engineering Inha University, Inchon, Korea
iHOP iHOP (Information Hyperlinked over Proteins). Protein association network built by literature mining
IntAct Protein interaction database at EBI. No species restriction.
InterDom Database of putative interacting protein domains. Institute for InfoComm Research, Singapore.
JCB PPI site at the Jena Centre for Bioinformatics, Germany
MetaCore Commercial software suite and database. Manually curated human PPIs
MINT Molecular INTeraction database at the Centro di Bioinformatica Moleculare, Universita di Roma, Italy.
MRC PPI links
Commented list of links to PPI databases and resources maintained at the MRC Rosalind Franklin Centre, Cambridge, UK
OPHID The Online Predicted Human Interaction Database. Ontario Cancer Institute and University of Toronto, Canada.
PDZbase Database of PDZ mediated protein-protein interactions.
Predictome Predicted functional associations and interactions. Boston University.
PathCalling Proteomics and PPI tool/database. CuraGen Corporation.
PIM Hybrigenics PPI data and tool, H. pylori. Free academic license available
RIKEN Experimental and literature PPIs in mouse.
STRING Protein networks based on experimental data and predictions at EMBL.
YPD "BioKnowledge Library" at Incyte Corporation.
Bioinformatic Approaches
http://dip.doe-mbi.ucla.edu/
Bioinformatic Approaches
http://mips.gsf.de/genre/proj/mpact/yeast/query/interaction
Bioinformatic Approaches
http://www.thebiogrid.org/
Bioinformatic Approaches
http://www.himap.org/main/main.jsp
Bioinformatic Approaches
http://www.ihop-net.org/UniPub/iHOP/
Quanternary Organization: A Case Study of PPIs
1º 2º 3º 4º
The workshop focuses not in the proteins themselves but in their
homo-oligomerization state as a tool to study the field of PPI and a
new methodology to approach this field.
15-Lipoxygenase
A 94-kDa protein with iron binding site. Three alternatively spliced transcript variants encoding two distinct isoforms have been described.
Activity: Arachidonate + O2 → 15S-hydroperoxyeicosatetraenoic acid
Function: Arachidonic acid metabolism & linoleic acid metabolism.
Interactions: Unknown.
Oligomerization: Monomer.
Beta-Enolase
A 47-kDa protein with magnesium binding site (required for catalysis and dimerization). Three isozyme subunits (alpha, beta, gamma) are known.
Activity: 2-phospho-D-glycerate → 2-phosphoenolpyruvate + H2O
Oligomerization: Homodimers or heterodimers.
Function: Glycolysis, Muscle development and regeneration.
Interactions: AChE.
Pyruvate Kinase Muscle Isozyme
A 57-kDa protein with four metal binding sites (magnesium and potassium). Three alternatively spliced transcript variants encoding two distinct isoforms have been reported and four mammalian isozymes: L (liver), R (red cells), M1 (muscle, heart and brain) and M2 (early fetal tissues).
Activity: ADP + 2-phosphoenolpyruvate → ATP + pyruvate
Oligomerization: Homotetramer; On subunit dissociation, a dimeric intermediate is formed.
Function: Glycolytic pathway.
Interactions: Thyroid hormone & Opa protein.
PK Oligomerization
The tetramer:dimer ratio of PK is not constant, and determines whether glucose carbons are converted to energy (tetrameric) or into synthetic processes (dimeric).
Oxygen starvation or highly accumulated glycolytic intermediates, such as fructose 1,6-bisphosphate or the amino acid serine, induce the reassociation of the dimeric form to the tetrameric form.
PEP
pyruvate
ADP
ATP
Ser/ fructose 1,6-P2